Frequently it is necessary to label molecules in order to provide a means of detecting and measuring the presence or absence of molecular entities, especially for the detection of biological molecules, chemical species and even certain types of organisms or cells. One of the trends to improve efficiency of identifying and quantifying these molecular or cellular entities is to perform multiplex types of analyses where the objective is to measure many of these species in a single assay such as microarrays or flow cytometric assays.
Presently RNA species within a specimen are isolated and usually converted to their respective cDNAs for analysis by microarrays, quantitative real-time RT-PCR and other methods well known in the art. Many of these methods are time consuming, and suffer from sample losses during the various isolation and repeated purification steps required to be performed to obtain a result. In particular many of the labeling agents presently used are very reactive and prone to decomposition especially in aquous solutions and substantial excess of labeling agents relative to the species to be labeled are frequently required for successful labeling. This is particularly the case for N-hydroxysuccinimide esters which are well known to be labile in aquous media and have half-lives of only a few minutes. More importantly, these esters generally are used to modify primary amines, consequently if target substances lack this functionality they must me modified to provide a free amine, thus adding additional steps to the labeling process and usually requiring additional steps of purification leading to loss of some sample. Likewise maleimides also suffer the same disadvantages with respect to their reactions with sulfhydryl groups. For example, methods presently described in the art rely on sequential reaction of the RNA or geminal diol containing species such as polysaccharides or glycosylated proteins or peptides with periodate, usually neutralization of the periodate with glycerol followed by separation of the oxidized RNA or resulting geminal aldehyde species of interest from the periodate to prevent reaction of periodate with the labeling moiety by for example ethanol precipitation, exclusion chromatography and the like. The isolated oxidized RNA or geminal aldehyde reaction product is then reacted with a hydrazide containing signal moiety and a second physical separation of the labeled RNA or geminal aldehyde species is performed such as ethanol precipitation, or chromatography. Because periodate is usually employed in the periodate reaction of geminal hydroxyl groups, the scavenging of periodate with glycerol is ineffective in removing the periodate, merely diverting the reaction to another species yet introducing promiscuous dialdehydes to the reaction which can consume significant portions of the labeling agent with consequent diversion to unproductive labeling of irrelevant species, namely the geminal aldehydes arising from the oxidation of glycerol or other geminal diol containing scavengers. There is a need for rapid labeling methods without these limitations.